Xterra rp c18 column

In vitro oxaliplatin release and % EE were determined by using HPLC Agilent 1200 Infinity Series HPLC system (Agilent Technologies, Waldbronn, Germany). Briefly, XTerra™ RPC₁₈ column (particle size 5 μm, inside diameter 4.6 mm, and length 250 mm; Waters Corporation, Milford, MA, USA) at 210 nm using an HPLC-UV spectrometer (Agilent 1290 infinity). The mobile phase was a 20:80 mixture of ACN and deionized water and was set to a flow rate of 0.8 mL/min (Model 1260 Quat Pump VL). The samples were diluted as necessary, and 20 μL of each sample was injected using an autosampler (Model 1260 ALS).2.9. Statistical Analysis
In vitro percent (%) cumulative oxaliplatin release and in vivo pharmacokinetic study data are expressed as the means ± standard deviations. The t-test or two-sided RM ANOVA and Bonferroni test were applied to the analyses of the differences between the groups. A p value < 0.05 was considered as a statistically significant difference.

The same HPLC-mass spectrometry system mentioned above was used in the targeted phenolic compound detection, and this time an Xterra RP-C18 column (Waters Corporation, Milford, MA, USA) with the dimensions of 3.9 × 100 mm and a particle size of 3.5 μm was used for optimal separation. Extracted bacterial samples were injected into the column for gradient elution separation at 0.900 mL/min using solvents A (89.9% water/10% acetonitrile/0.1% formic acid, v/v/v) and B (69.9% water/30% acetonitrile/0.1% formic acid, v/v/v). The autosampler temperature was kept at 4 °C, the column compartment was set at 30 °C, and the separation time for each sample was 11 min. The retention time and selected reaction monitoring (SRM) transitions of targeted metabolites and polyphenols (or compound pairs) were established by running pure standards and collecting the MS/MS data, and therefore, the orthogonal information on the retention time and two pairs of SRM transitions can be used to confidently detect and identify phenolic compounds. When biological samples were run, pooled polyphenol standards as quality controls were also tested in between every 10 samples to monitor the instrument stability.

Serotonin content was detected as published method (Jin et al., 2015). 200 mg leaf tissue at the heading stage was ground with liquid nitrogen into a powder and soaked in 1 mL 100% methanol. The homogenates were centrifuged at 12 000 g for 30 min and the supernatant was filtered through a syringe with a 0.22 μm PVDF membrane (Millipore). Then the filtrate was evaporated to dryness under refrigerating vacuum pump and dissolved in 500 μL 50% methanol. The samples were separated on an XTerra RP C18 column (250 × 4.6 mm, 5 μm, Waters) with an isocratic elution of 50% methanol in water containing 0.3% trifluoroacetic acid at a flow rate of 0.4 mL/min. A UV wavelength of 280 nm was used for detection.

For each sample, 100 mg leaf tissue was ground with liquid nitrogen into a powder and soaked in 2 ml 100% methanol. The homogenates were centrifuged at 10,000 × g for 10 min and the supernatant was filtered through a syringe with a 0.2 μm cellulose acetate membrane filter (Pall, USA). Then the filtrate was evaporated to dryness under vacuum and dissolved in 500 μl 50% methanol. The final sample was analyzed by reversed-phase HPLC (Waters, USA) so that the tryptophan and serotonin contents could be quantified. The samples were separated on an XTerra RP C18 column (250 × 4.6 mm, 5 μm, Waters) with an isocratic elution of 50% methanol in water containing 0.3% trifluoroacetic acid at a flow rate of 0.4 ml/min. A UV wavelength of 280 nm was used for detection. The standard samples for tryptophan and serotonin were made by Sigma (USA).

The chemical stability of thiamine in solution was measured in accordance with an adaptation of AOAC method 942.23 for quantification of thiamine [29 ]. Reverse-phase HPLC (Waters Corp. Milford, MA) using a gradient method with 0.1% TFA in water (v/v) and acetonitrile as the mobile phases, A and B, respectively, was used in accordance with our previous study [16 (link)]. Briefly, a Waters 2690 Separations Module and a Waters 2996 Photodiode Array (PDA) detector were used with a Waters XTerra RP-C₁₈ column and a wavelength scan of 235–400 nm. The gradient method was as follows: 100/0 at 0 min, 97/3 at 4 min (linear), 90/10 at 6 min (linear), 100/0 at 10 min (linear), and 100/0 at 15 min. Prior to analysis, solutions were cooled in an ice bath, and diluted with the 0.1% TFA in water mobile phase to an estimated thiamine concentration of 500 ppm, or 0.5 mg/mL (assuming no degradation). Standard curves of TMN and TClHCl (R² > 0.999) were prepared using the area under the analyte peak to calculate thiamine concentration of samples on each day of analysis using a concentration range of 10 ppm to 1000 ppm. Integration of the analyte peak was performed at 254 nm.